Attenuated live viruses, including genetically engineered recombinants, are known as useful vaccine vectors. The genome of the live virus can be engineered to carry genes encoding heterologous antigens against which immunological responses are desired in such a way that the replicative ability of the live virus is preserved, and that the heterologous gene is expressed in cells infected by the recombinant virus. The expressed antigens are thus available to provoke a useful immune response. The heterologous antigens may originate from an infectious pathogen, in order that a protective or therapeutic immune response can be mounted against the infectious agent, but alternatively they may represent tumour cell-specific or tumour-associated antigens; here the aim is to induce an immune response against tumour cells, to induce tumour rejection or regression.
More generally, recombinant viral vectors are among several known agents available for the introduction of foreign genes into cells so that they can be expressed as protein. A central element is the target gene itself under the control of a suitable promoter sequence that can function in the cell to be transduced. Known techniques include non-viral methods, such as simple addition of the target gene construct as free DNA; incubation with complexes of target DNA and specific proteins designed for uptake of the DNA into the target cell; and incubation with target DNA encapsulated e.g. in liposomes or other lipid-based transfection agents.
A further option is the use of recombinant virus vectors engineered to contain the required target gene, and able to infect the target cells and hence carry into the cell the target gene in a form that can be expressed. A number of different viruses have been used for this purpose including retroviruses, adenoviruses, and adeno-associated viruses.
Specification EP 0 176 170 (Institut Merieux: B Roizman) describes foreign genese inserted into a herpes simplex viral genome under the control of promote- regulatory regions of the genome, thus providing a vector for the expression of the foreign gene. DNA constructs, plasmid vectors containing the constructs useful for expression of the foreign gene, recombinant viruses produced with the vector, and associated methods are disclosed.
Specifications EP 0 448 650 (General Hospital Corporation: AI Geller, XO Breakefield) describes herpes simplex virus type 1 expression vectors capable of infecting and being propagated in a non-mitotic cell, and for use in treatment or neurological diseases, and to produce animal and in vitro models of such diseases.
Recombinant viruses are known in particular for use in gene therapy applied to gene deficiency conditions.
Examples of genes used or proposed to be used in gene therapy include: the gene for human adenosine deaminase (ADA), as mentioned in for example WO 92/10564 (KW Culver et al: US Secretary for Commerce & Cellco Inc), WO 89/12109 & EP 0 420 911 (IH Pastan et al); the cystic fibrosis gene and variants described in WO 91/02796 (L-C Tsui et al: HSC Research & University of Michigan), in WO 92/05273 (FS Collins & JM Wilson: University of Michigan) and in WO 94/12649 (RJ Gregory et al: Genzyme Corp).
The prior art of malignant tumour treatment includes studies that have highlighted the potential for therapeutic vaccination against tumours using autologous material derived from a patient's own tumour. The general theory behind this approach is that tumour cells may express one or more proteins or other biological macromolecules that are distinct from normal healthy cells, and which might therefore be used to target an immune response to recognise and destroy the tumour cells.
These tumour targets may be present ubiquitously in tumours of a certain type. A good example of this in cervical cancer, where the great majority of tumours express the human papillomavirus E6 E7 proteins. In this case the tumour target is not a self protein, and hence its potential as a unique tumour-specific marker for cancer immunotherapy is clear.
There is increasing evidence that certain self proteins can also be used as tumour target antigens. This is based on the observation that they are expressed consistently in tumour cells, but not in normal healthy cells. Examples of these include the MAGE family of proteins. It is expected that more self proteins useful as tumour targets remain to be identified.
Tumour associated antigens and their role in the immunobiology of certain cancers are discussed for example by P van der Bruggen et al, in Current Opinion in Immunology, 4(5) (1992) 608-612. Other such antigens, of the MAGE series, are identified in T. Boon, Adv Cancer Res 58 (1992) pp 177-210, MZ2-E and other related tumour antigens are identified in P. van der Bruggen et al, Science 254 (1991) 1643-1647; tumour associated mucins are mentioned in PO Livingston, in current Opinion in Immunology 4 (5) (1992) pp 624-629; e.g. MUCl as mentioned in J Burchell et al, Int J Cancer 44 (1989) pp 691-696.
Although some potentially useful tumour-specific markers have thus been identified and characterised, the search for new and perhaps more specific markers is laborious and time-consuming, and with no guarantee of success.
Administration to mammals of cytokines as such has been tried, but is often poorly tolerated by the host and is frequently associated with a number of side-effects including nausea, bone pain and fever. (A Mire-Sluis, TIBTech vol. 11 (1993); MS Moore, in Ann Rev Immunol 9 (1991) 159-91). These problems are exacerbated by the dose levels after required to maintain effective plasma concentrations.
Virus vectors have been proposed for use in cancer immunotherapy to provide a means for enhancing tumour immunoresponsiveness.
It is known to modify live virus vectors to contain genes encoding a cytokine or tumour antigen: see specification WO 94/16716 (E Paoletti et al: Virogentics Corp.) and reference cited therein: WO 94/16716 describes, for use in cancer therapy, attenuated recombinant vaccinia viruses containing DNA coding for a cytokine or a tumour antigen. Cytokines are examples of immunomodulating proteins. Immuno-modulating proteins which enhance the immune response such as the cytokine, interleukin 1, interleukin 2 and granulocyte-macrophage colony stimulating factor (GM-CSF) (see for example A W Heath et al., Vaccine 10 (7) (1992), and Tao Mi-Hua et al., Nature 362 (1993)), can be effective vaccine adjuvants.
It has been proposed to use GMCSF-transduced tumour cells as a therapeutic vaccine against renal cancer. The protocols for corresponding trials involve removal of tumour material from the patients, and then transduction with the appropriate immunomodulator gene. The engineered cells are then to be re-introduced into the patient to stimulate a beneficial immune response.
Although it has been proposed to introduce immunomodulatory genes into certain kinds of tumour cells, existing methods are considered to have limitations, whether the difficulties are due to low quantitative amounts of transduction, to complexity, or to undesirable side-effects of the systems employed.
Recently, an experimental intracranial murine melanoma has been described as treated with a neuroattenuated HSV1 mutant 1716 (BP Randazzo et al., Virology 211 (1995) pp94-101), of which the replication appeared to be restricted to tumour cells and not to occur on surrounding brain tissue.
Furthermore, vectors based on herpesvirus saimiri, a virus of non-human primates, have been described as leading to gene expression in human lymphoid cells (B Fleckenstein & R Grassmann, Gene 102(2) (1991), pp 265-9). However, it has been considered undesirable to use such vectors in a clinical setting.
The prior art includes specification WO 92/05263 (Inglis et al: Immunology Limited) (the content of which is incorporated herein by reference), which describes for example the use as vaccine of a mutant virus whose genome is defective in respect of a gene essential for the production of infectious virus, such that the virus can infect normal host cells and undergo replication and expression of viral antigen genes in such cells but cannot produce infectious virus. WO 92/05263 particularly describes an HSV virus which is disabled by the deletion of a gene encoding the essential glycoprotein H (gH) which is required for virus infectivity (A Forrester et al, J Virol 66 (1992) 341-348). In the absence of gH protein expression non-infectious virus particles providing almost the complete repertoire of viral proteins are produced. These progeny virus, however, are not able to infect host cells and spread of the virus within the host is prevented. Such a virus has been shown to be an effective vaccine in animal model systems (Farrell et al, J Virol 68 (1994) 927-932; McLean et al, J Infect Dis, 170 (1994) 1100-9). These mutants viruses can be cultured in a cell line which expresses the gene product in respect of which the mutant virus is defective. Cell lines which are suitable for the culture of certain viruses of this type have been described in the literature: for example in references given in cited specification WO 92/05263.
Complete or substantial sequence data has been published for several viruses such as Epstein-Barr virus EBV (Baer et al, in Nature 310 (1984) 207), human cytomegalovirus CMV (Weston and Barrell in J Mol Biol 192 (1986) 177-208), varicella zoster virus VZV (Davison and Scott, in J Gen Virol 67 (1986) 759-816) and herpes simplex virus HSV (McGeoch et al, in J. Gen. Virol. 69 (1988) 1531-1574). The gH glycoprotein is known to have homologues in EBV, CMV and VZV (Desai et al, in J Gen Virol 69 (1988) 1147).
Virus vectors provide an opportunity for intracellular delivery of both DNA and protein, for immunisation and gene therapy, e.g. corrective gene therapy, as well as for use in for example cancer immunotherapy, but the prior art leaves it still desirable to provide further viral vectors and processes useful for transforming human and non-human animal cells and expressing proteins therein.